HK1121403A1 - Molecules that are able to inhibit the binding between ngf and the trka receptor as analgesics with prolonged effect - Google Patents

Abstract

Use of an anti-NGF antibody capable of inhibiting the binding between NGF and TrkA, capable of blocking the biological activity of TrkA for the preparation of a medicament for treating and/or preventing chronic pain.

Description

Molecules capable of inhibiting NGF binding to TrkA receptors as long-acting analgesics

Background

The present invention relates to the use of molecules capable of inhibiting the binding between NGF and its receptor TrkA. In particular, the invention relates to antibodies to one of these two molecules, which have a long-term analgesic effect by blocking the biological activity of NGF. Due to their long-term analgesic effect, they can be advantageously used for the treatment of persistent pain (also known as chronic pain), such as but not limited to neuropathic pain or neoplastic pain.

Prior Art

Nociceptive signaling is transmitted into the spinal cord via a δ and C fibers, the soma (primary sensory neurons) of which are located in the spinal cord dorsal ganglia (DRGs). Primary sensory neurons release glutamate and ATP as excitatory neurotransmitters and release many other substances such as substance P and CGRP (calcitonin gene-related peptide) (Hunt and Mantyh, 2001). The release of these excitatory neurotransmitters is controlled by various types of receptors present on afferent terminals, including capsaicin-sensitive receptors (vanilloid receptor, VR1), receptors activated by GABA, receptors activated by ATP itself, and receptors activated by cannabinoids (CB1) (Sivilotti and Nistri, 1991; Hunt and Mantyh, 2001; Khakh, 2001; Morisset et al, 2001). One pathological mechanism for the development of chronic pain is allodynia (allodynia), i.e. the conversion of a normally pain-free stimulus into a painful sensation. This phenomenon involves a variety of ionic currents and hence a variety of "ligand-gated" type channels, including the capsaicin receptor VR1 and the ionotropic receptor for ATP (Khakh, 2001). The simultaneous activation of VR1 receptors and ATP receptors on spinal cord injury interneurons produces a massive accumulation of excitatory synaptic signals that enhances the transmission of painful stimuli (Nakatsuka et al, 2002). Based on this, it is therefore clear that ATP receptors (in particular those belonging to the P2X3 type) play a major role in the pain pathway (Burnstock, 2001). These receptors are present in peripheral nerve terminals activated by pain stimuli, in neuronal cell bodies and their presynaptic terminals in DRGs, and naturally on postsynaptic terminals of the spinal cord (Khakh, 2001). There is a great deal of evidence that the system consisting of Nerve Growth Factor (NGF) and its high affinity receptor TrkA (Levi-Montalcini, 1987; Levi-Montalcini et al, 1996; Frade and Barde, 1998; Kaplan, 1998) plays a major role in the molecular processes of the major form of "sustained" pain. This suggests a major therapeutic area for antibodies that block the NGF/TrkA system (one of the pains, in particular "stressful" pain) (Levine, 1998). The development of sensory nociceptive neurons depends largely on NGF, and the response of adult nociceptors is modulated by this factor (Julius and Basbaum, 2001). In particular, NGF exerts a sensitizing capsaicin pain stimulus (Shu and Mendell, 1999). From a functional point of view, the frequency and duration of the action potential of injured neurons after chronic inflammation change. These phenomena are restored by blocking endogenous NGF so that the hyperexcitability typically possessed by chronic pain states is markedly attenuated (Djouhri et al, 2001). The NGF actions that define pain thresholds in adult nociceptors are mediated by the TrkA receptor and also by responses mediated by VR1 receptors present in nociceptive terminals. The TrkA-dependent enhancement of VR1 response is thought to occur via the intracellular transduction pathway of the gamma form of phospholipase C (PLC γ, Chuang et al, 2001). Peripheral NGF levels are elevated during inflammation, while exogenous administration of NGF has hyperalgesic effects in rats and produces muscle pain in humans. In addition, NGF is often hypersensitive to thermal stimuli in humans and mammals. NGF is released by mast cells, fibroblasts and other cell types at the peripheral site where inflammation occurs. In particular, it appears that mast cells play a major role (Woolfet al., 1996). Since these cells produce NGF while expressing functional TrkA receptors on their surface (Nilsson et al, 1997), they are able to respond to NGF itself in the presence of lysophosphatidylserine (Horigome et al, 1993; Kawamoto et al, 2002). As a result, it appears that the NGF/TrkA system mediates mast cell activation through a positive feedback autocrine mechanism that allows local amplification of painful inflammatory signals. High levels of NGF are also found in neurons where this neurotrophin is apparently associated with modifications of nerve fibers associated with pain (Harpf et al, 2002). In certain forms of cancer, excess NGF promotes the growth and infiltration of nerve fibers to induce cancer-associated pain (Zhu et al, 1999). Recent experimental studies have shown that neuroma formation associated with neuropathic pain (neuropathic pain) can be significantly reduced by blocking NGF without destroying damaged neuronal cell bodies (Kryger et al, 2001). These results greatly motivate interest in therapeutic approaches to treat chronic pain based on reduced NGF effects (saragovian Gehring, 2000). In recent years, it has also been demonstrated, based on genetics, that the NGF/TrkA system is involved in the molecular processes of pain transduction. In particular, mutations in the TrkA gene (mutations located on chromosomes 1q21-q 22) are associated with an autosomal recessive genetic syndrome known as CIPA (congenital pain insensitivity with anhidrosis) which is characterized by periodic transient fever, anhidrosis, unresponsiveness to stimuli that cause pain, mental retardation and propensity to self-mutilation (Indo et al, 1996; Saragovi and Gehring, 2000; Indo, 2001; hido et al, 2001). The involvement of NGF in nociceptive responses was further confirmed recently by the results of phenotypic characterization of anti-NGF transgenic mice (AD 11). In these animals, ectopic expression of anti-NGF antibody α D11 resulted in blockade of NGF function in adults. This blockade translates in a consistent manner into an increase in latency in response to noxious thermal stimuli (Capsoni et al, 2000; Ruberti et al, 2000). Antibodies that neutralize the biological activity of the NGF/TrkA system by blocking the ligand or receptor may be an important tool in the treatment of pain, particularly in the treatment of persistent pain. In this context, recent publications indicate a significant reduction in pain with neutralizing anti-NGF antibodies for treatment in a mouse model of neoplastic pain (Sevcik et al, 2005). However, in the dosing regimen used by Sevcik et al, the maximum time delay between the last injection of anti-NGF and the observed results was not more than 4 days, and therefore it was not a long-term effect.

By long-term effect is meant an effect that is clearly sustained for at least 1-2 weeks after the last administration of the antibody, suggesting that there is no necessary correlation between the effect and the blood flow concentration of the antibody itself. Long-term effects may require new gene expression, possibly representing a sustained or prolonged modification of the original pathophysiological state. In many cases, drugs that produce long-term effects may be referred to as "disease-modifying" active ingredients, i.e., capable of profoundly altering the disease process, unlike products that exhibit only a simple pharmacological effect on the symptoms.

The inventors have studied a group of antibodies (antibodies directed against NGF ligands) capable of blocking the biological effect of NGF mediated by TrkA ligands. Two agents are of particular interest: α D11 (anti-NGF) and MNAC13 (anti-TrkA). The contrast between two antibodies (one directed to the ligand and the other to the receptor) is also of particular interest, since inhibition of NGF ligand is not functionally equivalent to inhibition of TrkA receptor. In fact three points must be considered:

i) for stoichiometric reasons, the availability of ligands and receptors may vary significantly and in different ways over time in the same system;

ii) the presence of a second receptor for NGF (p75) common to all neurotrophins and mediating a biological function different from that of TrkA (Hempstead, 2002);

iii natural occurrence of NGF in the "immature" form (pre-pro-NGF), characterised by unique properties in terms of biological activity and preferential binding to the p75 receptor (Lee et al, 2001).

α D11 is a rat monoclonal antibody directed against mouse NGF (but also recognises both rat and human NGF). Its interaction with NGF inhibits its binding to TrkA, blocking its physiological effects (cottaneo et al, 1988). α D11 also inhibits NGF binding to the p75 receptor. This anti-NGF antibody is absolutely unique in its specificity of binding to its antigen (in contrast to all other neurotrophins), characterized by a binding affinity (picomolar) to the antigen and by neutralizing features shown both in vitro and in vivo (Cattaneo et al, 1988; Berardi et al, 1994; Molnar et al, 1997; Molnar et al, 1998). The α D11 epitopes are located at the NGF loop I and/or NGF loop II levels exposed on the outside of the molecule and in close spatial proximity to each other. In addition, the conserved reactivity of α D11 in different species is consistent with the epitope arrangement (epitopic) because the amino acid residues of these two loops are highly conserved. The potent neutralizing activity of α D11 shows that the recognized epitope is very close to the NGF receptor binding site. In addition, the lack of cross-reactivity of α D11 with other members of the neurotrophin family suggests that: i) the epitope is located in an NGF region not shared with other neurotrophins, ii) the epitope itself may be involved in a "specific pathway" mediating NGF-TrkA recognition. The epitope recognized by the α D11 antibody on NGF molecules was identified by testing the binding activity of the antibody to a wide range of NGF mutants. Based on this systematic screen, a region of the NGF molecule (amino acids 41-49, loop I) was identified that is highly expressed in the upper part of the NGF molecule and is associated (although not exclusively) with the binding of this antibody to its antigen (Gonfloni, 1995). In fact, the region of amino acids 23-35 of NGF (Loop II) also contributes to binding.

The antibody MNAC13 is a mouse monoclonal antibody directed against the human TrkA receptor (Cattaneo et al, 1999; Pesavento et al, 2000), which is particularly effective in inhibiting the NGF activation process and downstream biological functions of TrkA in vitro and in vivo (Cattaneo et al, 1999; Pesavento et al, 2000). The antibodies are characterized by their structure (Covaceuszach et al, 2001) and molecular interaction with TrkA receptors (Covaceuszach et al, 2005).

Based on this insight into the structure by the innovative approach, humanized versions of both α D11 and MNAC13 antibodies (Hu- α D11 and Hu-MNAC13) were generated, showing the same antigen binding characteristics as the parent (patent application WO 05/061540).

It has been found that currently available treatments for neuropathic pain (caused by primary damage or by neurological dysfunction, such as pain associated with spinal cord injury), neoplastic pain, and numerous other forms of persistent pain (inflammatory nature) are of limited effectiveness. Therefore, there is a clear need to identify and reveal new molecules with analgesic activity and acting through a different mechanism of action than the commonly used analgesic drugs to solve the problems associated with side effects. International patent application WO02/20479 discloses small synthetic molecules that inhibit the TrkA receptor and have potent analgesic activity. However, the effect of these molecules on certain pain models has not been demonstrated. In addition, the small molecules have the disadvantage of being more likely to cross the blood brain barrier than antibodies, which may lead to serious side effects. Indeed, cholinergic neurons of the basal forebrain, a group of neurons affected by various forms of progressive neurodegeneration including alzheimer's disease (Saper et al, 1985), express the TrkA receptor and rely on NGF for proper function (Holtzman et al, 1992). International patent application WO 01/78698 proposes the use of NGF antagonists for the prevention or treatment of persistent visceral pain, but not for neuropathic or neoplastic pain. Even though the application states that the antagonist binds both NGF and TrkA receptors, it does not demonstrate that the receptor is functionally blocked when the antagonist binds to TrkA receptor. Based on the ability of the two antibodies MNAC13 and α D11 to block NGF/TrkA biological activity, the two antibodies MNAC13 and α D11 and their respective humanized forms were tested in various (rodent) animal models of persistent pain, in particular in the CCI model (chronic compressive injury, chronic compressive injury of the sciatic nerve), one of the models that can be used to evaluate chronic neuropathic pain (Bennett and Xie, 1988).

Summary of The Invention

The invention relates to the use of an anti-NGF antibody capable of inhibiting the binding of NGF to TrkA in the preparation of a medicament for the treatment of chronic pain.

An anti-NGF molecule that blocks the biological activity of TrkA is defined as a molecule that acts as an antagonist of NGF binding to TrkA receptors and comprises a synthetic molecule or a monoclonal antibody or a biological/synthetic derivative thereof which:

i) binding to TrkA;

ii) inhibits NGF binding to "native" TrkA receptors expressed on the surface of living cells ("native" means "native conformation in vivo"); and

iii) blocking the biological activity of NGF derived from binding to the same TrkA receptor.

The term "blocking biological activity" refers not only to blocking the activation of a receptor, i.e. to blocking the process of conversion of the receptor itself into the "active" state, but also to functional neutralization of the biological consequences downstream of this activation process, i.e. second messengers, new gene expression, phenotypic and functional changes. The molecule not only blocks TrkA in vitro tests (nerve growth tests in PC12 cells), but also blocks TrkA in vivo (functional blockade of cholinergic neurons of the basal forebrain and blockade of nociception in classical "hot plate" tests).

An object of the present invention is the use of an anti-NGF antibody capable of inhibiting the binding of NGF to TrkA in the preparation of a medicament for the treatment and/or prevention of chronic pain. Preferably, the antibody recognizes and binds NGF molecular domains comprising amino acid region EVNINNSVF (SEQ ID No.9) 41-49 of human or rat NGF, more preferably the domain comprising amino acid region GDKTTATDIKGKE (SEQ ID No.10) 23-35. More preferably, the antibody blocks the biological activity of TrkA.

In another aspect, the invention provides a method of treating and/or preventing chronic pain in a subject, comprising administering to the subject an effective amount of an anti-NGF antibody, thereby treating and/or preventing chronic pain in the subject. The invention also provides a kit comprising a composition comprising an anti-NGF antibody and instructions for administering the composition to a subject in need of treatment and/or prevention of chronic pain, thereby treating or/preventing chronic pain in the subject.

In a preferred aspect, the variable region of the antibody light chain comprises at least one, more preferably two, most preferably three Complementarity Determining Regions (CDRs) having a sequence selected from amino acids 24-34 of SEQ ID No.1, amino acids 50-56 of SEQ ID No.1, amino acids 89-97 of SEQ ID No. 1.

In another preferred aspect, the variable region of the antibody light chain substantially comprises the sequence of SEQ ID No. 1.

(VL，SEQ ED No.1)：

L CDR1 L CDR2

DIQMTQSPASLSASLGETVTIECRASEDIYNALAWYQQKPGKSPQLLIYNTDTLHTGVP

L CDR3

SRFSGSGSGTQYSLKINSLQSEDVASYFCQHYFHYPRTFGGGTKLELK

In a preferred aspect, the antibody heavy chain variable region comprises at least one, more preferably two, most preferably three Complementarity Determining Regions (CDRs) having a sequence selected from amino acids 26-35 of SEQ ID No.2, amino acids 50-65 of SEQ ID No.2, and amino acids 98-111 of SEQ ID No. 2.

In another preferred aspect, the antibody heavy chain variable region substantially comprises the sequence of SEQ ED No. 2.

(VH，SEQ ID NO 2)：

H CDR1 H CDE2

QVQLKESGPGLVQPSQTLSLTCTVSGFSLTNNNVNWVRQATGRGLEWMGGVWAGGATDY

H CDR3

NSALKSRLTITRDTSKSQVFLKMHSLQSEDTATYYCARDGGYSSSTLYAMDAWGQGTTV

TVSA

The antibody may be in single chain form and comprise a light chain variable region and a heavy chain variable region linked by a linker.

Alternatively, the antibody may comprise two light chains and two heavy chains.

In a preferred aspect of the invention, the anti-NGF antibody is a human antibody or a humanized antibody. The skilled person will select a suitable humanisation method to design an antibody, a preferred method being that disclosed in WO 2005/061540.

Briefly, "humanized" variants of the antibody variable regions are obtained by grafting the Complementarity Determining Regions (CDRs) of a rat antibody onto human immunoglobulin frameworks. The complete structural information from X-ray diffraction studies on Fab fragments of the α D11 antibody was used to select acceptor frameworks of human origin. Two different criteria were used to minimize the structural differences between the rat α D11 antibody and the recipient human antibody: i) level of primary structural homology, ii) level of three-dimensional structural similarity. Following selection of the framework, substitution of the human residues by the rat counterpart was minimized to reduce the potential immunogenicity of the resulting humanized antibody.

An exemplary humanized antibody comprises a light chain variable region which is the amino acid sequence of SEQ ID No: 1 (rat-derived sequence). An exemplary humanized antibody comprises a heavy chain variable region which is the amino acid sequence of SEQ ID No: 2 (rat-derived sequence).

In a preferred aspect of the invention, the variable region of the humanized antibody light chain substantially comprises the sequence shown in SEQ ID No. 3.

SEQ ID NO: 3(VL, light chain variable region of Hu- α D11):

L CDR1 L CDR2

DIQMTQSPSSLSASVGDRVTITCRASEDIYNALAWYQQKPGKAPKLLIYNTDTLHTGVP

L CDR3

SRFSGSGSGTDYTLTISSLQPEDFATYFCQHYFHYPRTFGQGTKVEIK

in a preferred aspect of the invention, the variable region of the heavy chain of the humanized antibody comprises substantially the sequence shown in SEQ ID No. 4.

SEQ ID No.4(VH, heavy chain variable region of Hu- α D11):

H CDR1 H CDR2

EVOLVESGGGLVQPGGSLRLSCAASGFSLTNNNVNWVRQAPGKGLEWVGGVWAGGATDY

H CDR3

NSALKSRFTISRDNSKNTAYLQMNSLRAEDTAVYYCARDGGYSSSTLYAMDAWGQGTLV

the humanized variable regions described above were cloned into a suitable expression vector in the form of human IgG1 or IgG4 isotype and transfected into mammalian cell lines for expression, purification and pharmaceutical characterization.

Different variants of Hu- α D11 (complete IgG: heavy chain + light chain) were finally generated (different due to different constant regions).

In a preferred aspect of the invention, the humanized antibody light chain has substantially the sequence shown in SEQ ID No. 8.

SEQ ID NO 8, Hu-alpha D11Vk human Ck

DIQMTQSPSSLSASVGDRVTITCRASEDIYNALAWYQQKPGKAPKLLIYNTDTLHTGVP

SRFSGSGSGTDYTLTISSLQPEDFATYFCQHYFHYPRTFGQGTKVEIKRTVAAPSVFIF

PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

(variable regions in italics; mutations in the rat sequence during humanization; CDRs in underline).

In a preferred embodiment, the humanized anti-NGF antibody heavy chain has substantially one of the following three sequences:

SEQ ID NO 5, Hu-anti-NGF (VH) human IgG1

EVQLVESGGGLVQPGGSLRLSCAASGFSLTNNNVNWVRQAPGKGLEWVGGVWAGGATDY

NSALKSRFTISRDNSKNTAYLQMNSLRAEDTAVYYCARDGGYSSSTLYAMDAWGQGTLV

TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA

PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK

PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY

TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 6, Hu- α D11(VH) human IgG 1(IgG 1 with N297A mutation, as described by Bolt et al, 1993)

EVQLVESGGGLVQPGGSLRLSCAASGFSLTNNNVNWNRQAPGKGLEWVGGVWAGGATDY

NSALKSRFTISRDNSKNTAYLOMNSLRAEDTAVYYCARDGGYSSSTLYAMDAWGQGTLV

TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA

PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK

PREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY

TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 7, Hu-alpha D11(VH) human IgG4

EVQLVESGGGLVQPGGSLRLSCAASGFSLTNNNVNWVRQAPGKGLEWVGGVWAGGATDY

NSALKSRFTISRDNSKNTAYLQMNSLRAEDTAVYYCARDGGYSSSTLYAMDAWGQGTLV

TVSSASTKGPSVFPLAPSSKSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

VLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEF

LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE

EQFNSTYRWWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP

PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT

VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

(variable regions in italics; mutations in rat sequences during humanization; CDRs in underlining; N297A mutation eliminates the glycosylation site).

In a preferred aspect, the molecules of the invention are used for the preparation of a medicament for the treatment of chronic inflammatory type pain, preferably due to the following diseases: pancreatitis, kidney stones, headache, dysmenorrhea, musculoskeletal pain, sprain, visceral pain, ovarian cyst, prostatitis, cystitis, interstitial cystitis, post-operative pain, migraine, trigeminal neuralgia, pain from burns and/or wounds, pain associated with trauma, neuropathic pain, pain associated with musculoskeletal diseases, rheumatoid arthritis, osteoarthritis, ankylosing myelitis, periarticular lesions, neoplastic pain, bone metastatic pain, HIV pain.

Alternatively, the pain is neuropathic pain or neoplastic pain.

Pain is generally interpreted as "unpleasant sensory and emotional experiences associated with actual or potential tissue damage, or as unpleasant sensory and emotional experiences of such damage, or both," according to the International Association for the Study of pain, IASP, www.iasp-pain. The basic factors in all forms of pain are the activation of specialized high threshold receptors and nerve fibers alerting the organism to the presence of potential tissue damage. The involvement of inflammatory cells and processes is a common factor in many pain states. The term "acute pain" refers to immediate, usually high threshold, pain resulting from injury such as cutting, crushing, burning or from chemical stimulation. The term "chronic pain" as used herein refers to pain other than acute pain. It will be appreciated that chronic pain is typically of a long duration, for example months or years, and may be continuous or intermittent.

The anti-NGF antibody is suitably administered systemically. Systemic administration of anti-NGF antibodies can be by injection, e.g., continuous intravenous infusion, bolus intravenous injection, subcutaneous or intramuscular injection. Alternatively, other modes of administration (e.g., oral, transmucosal, inhalation, sublingual, etc.) may be used. Local administration of the antibody can be performed, for example, by intra-articular injection or subcutaneous, intramuscular injection in the vicinity of the affected tissue.

The anti-NGF antibodies are suitably formulated as pharmaceutical compositions suitable for the specified route of administration. The injection solution suitably also has the antibody dissolved or dispersed in a liquid medium (e.g. water for injection) containing a suitable buffer and a molarity modifier such as phosphate, salt and/or glucose.

The treatment regimen, i.e., dose, timing and repetition, can be administered once or repeatedly (e.g., by injection) by the chosen route of administration. The interval between doses administered may vary according to the degree and duration of clinical response, as well as the particular individual and the individual's clinical history. Suitably, the anti-NGF antibody has a longer duration of action. In particular, the clinical effect of the antibody was determined to last 21 days after administration according to animal studies. In addition, preliminary data suggest that the anti-NGF antibodies may present a longer lasting clinical benefit than if their presence was detected in the relevant biological matrix, such as serum or plasma, following administration.

Depending on the length of action specified (i.e. suitably for at least one week or preferably at least two weeks such as at least three weeks or at least four weeks), the antibody may suitably be administered to the subject not more frequently than once per week, for example not more frequently than once every two weeks or once every three weeks or once every four weeks.

Suitable doses of anti-NGF antibodies typically range from 0.1mg/kg to 10mg/kg body weight.

Novel antibodies and compositions containing the same are an aspect of the invention.

Non-limiting embodiments of the present invention will now be disclosed with particular reference to the following drawings.

FIG. 1: BIAcore analysis of the binding of α D11 anti-NGF antibodies to mouse NGF (m-NGF) and recombinant mouse pronNGF (rm-pronNGF). The α D11 anti-NGF antibody was immobilized on flow cell (flow cell)2, while flow cell 1 was blank. Each curve is obtained by subtracting the background signal (measured in cell 1) from the signal measured in cell 2. The surface plasmon resonance signal gives the amount of surface-bound components at each stage, expressed in Resonance Units (RU).

For m-NGF binding, the immobilized antibody was 3000 Resonance Units (RU) in experimental group A and 6000RU in experimental group B. The injected concentration of m-NGF is indicated above each curve. From a complete analysis of the data, the affinity parameters were evaluated, with the following results: KA 3.55 · 10¹¹l/M；KD＝2.81·10^-12M(chi²Value 0.123).

For rm-proNGF binding (group C), the immobilized antibody was 3000 RU. The injection concentration of rm-proNGF is indicated above each curve. Kinetic analysis of the data allows the following parameters to be evaluated: KA 1.2 · 10⁹l/M；KD＝1.9·10^-9M(chi²Value 0.09).

FIG. 2: effect of Fab α D11(α D11) and Fab Hu- α D11(Hu- α D11) anti-NGF antibodies on Formaldehyde-induced pain (phase 2 of Formaldehyde assay: 15-40 min. phase 2 corresponds to inflammation-related pain). The mice were injected subcutaneously on the back of the right hind paw with 5% formaldehyde.

Treatment included antibody injection for 45 minutes (Fab. alpha. D11 or Fab H)u- α D11 was injected into simulated Fab or saline) (in the same paw as the formaldehyde injection), followed by formaldehyde injection and tested (one injection of each antibody: 12.5. mu.g). Each experimental group included at least 8 animals. Statistical analysis of the data showed significant analgesic effects of anti-NGF treatment (both parental and humanized forms of the antibody), apparently specific for the second phase of the pain response (inflammation) (licking time). For saline group: (^**p < 0.01) or mock Fab treated group (# p < 0.05), the effects of anti-NGF antibodies (both parental and humanized forms) were statistically different (ANOVA).

FIG. 3: effect of anti-TrkA monoclonal antibody MNAC13(1.4mg/kg) and anti-NGF monoclonal antibody α D11(1.4mg/kg) on neuropathic pain: mechanical allodynia measured by plantar dynamic tactile meter; CD1 mice were subjected to chronic compression of the sciatic nerve and the antibodies were injected on days 3, 4, 5, and 6 after sciatic nerve injury. The observation period ranged from day 3 to day 14. Saline (sal) and mouse immunoglobulin (IgG, 1.4mg/kg) were used as negative controls. The results are expressed as absolute values (grams) of the pressure threshold of the hind paw ipsilateral to the injury. Statistical analysis of the data was performed using analysis of variance with repeat assays (ANOVA), where the "treatment" factor and repeat assay (days) were statistically significant and p < 0.01. Animals treated with either anti-TrkA or anti-NGF differed significantly from the control group from day 4 to day 14.

FIG. 4: effect of anti-TrkA monoclonal antibody MNAC13(1.4mg/kg) and anti-NGF α D11 antibody (1.4mg/kg) on neuropathic pain: mechanical touchdown was measured by a plantar dynamic tactile meter. CD1 mice were subjected to chronic compression of the sciatic nerve and were injected LP. days 3, 4, 5, 6 post sciatic nerve injury with the antibodies. The observation period ranged from day 3 to day 14. Saline (sal) and mouse immunoglobulin (IgG, 1.4mg/kg) were used as negative controls. Results are expressed as a percentage (% of hind paws ipsilateral to injury versus threshold pressure ratio corresponding to contralateral hind paws). Statistical analysis of the corresponding absolute values was performed using analysis of variance (ANOVA) for replicate assays, where the "treatment" factor and replicate assay (days) are statistically significant and p < 0.01 (at least). Animals treated with either anti-TrkA or anti-NGF differed significantly from the control group from day 4 to day 14.

FIG. 5: comparison of the effects of anti-TrkA monoclonal antibody MNAC13(2 doses: 0.9 and 2mg/kg) and anti-NGF monoclonal antibody α D11(2mg/kg dose) on neuropathic pain: mechanical touchdown was measured by a plantar dynamic tactile meter. CD1 mice were subjected to chronic compression of the sciatic nerve and the antibodies were injected i.p. on days 3, 4, 5, 6, 7, 8, 9, 10 after sciatic nerve injury. The observation period ranged from day 3 to day 31. Mouse immunoglobulin (IgG, 2mg/kg) was used as a negative control. Results are expressed as a percentage (% of hind paws ipsilateral to injury versus threshold pressure ratio corresponding to contralateral hind paws). Statistical analysis of the corresponding absolute values was performed using analysis of variance (ANOVA) for replicate assays, where the "treatment" factor and replicate assay (days) are statistically significant and p < 0.01 (at least). Animals treated with MNAC13 differed significantly from the control group from day 5 (higher dose of MNAC13) or from day 7 (lower dose of MNAC13) until the last day of observation (day 31). Animals treated with α D11 differed significantly from the control group from day 4 to day 14 and from day 21 to day 31 until the last day of observation (day 31).

FIG. 6: comparison of the effects of parent (. alpha.D 11) and humanized versions (Hu-. alpha.D 11, human IgG4 version) of anti-NGF neutralizing antibodies (test 1: 2mg/Kg) on neuropathic pain: measuring mechanical touch pain by a sole dynamic tactile meter; CD1 mice were subjected to CCI (chronic compressive injury) of the sciatic nerve and the antibodies were injected i.p. on days 3, 4, 5, 6, 7, 8, 9, 10 post sciatic nerve injury. The observation period ranged from day 3 to day 31. Rat immunoglobulin (IgG, 2mg/kg) was used as a negative control. Results are expressed as a percentage (% of hind paws ipsilateral to injury versus threshold pressure ratio corresponding to contralateral hind paws). Statistical analysis of the corresponding absolute values was performed using analysis of variance (ANOVA) for replicate assays, where the "treatment" factor and replicate assay (days) are statistically significant and p < 0.01 (at least). Animals treated with α D11 or Hu- α D11 differed significantly from the control group from day 4 to day 14 and from day 21 to day 31 until the last day of observation (day 31).

Method of producing a composite material

Production of monoclonal antibodies

Monoclonal antibodies MNAC13 and α D11 were produced from the hybridoma supernatants according to the standard procedures disclosed above (Galfre and Milstein, 1981; Cattaneo et al, 1988; Cattaneo et al, 1999). The supernatant containing each antibody was precipitated (29% ammonium sulfate), followed by dialysis against PBS IX (Spectra-Por 12/14K membrane, Spectrum) and affinity chromatography on an agarose protein G chromatography column (4-Fast Flow, Amersham Biosciences). Elution was performed with a low pH solution (HCl, 5mM) and neutralization was performed upon collection. The final eluate (Amicon Ultra-15, 50K, Millipore) was concentrated to obtain a purified antibody preparation at a concentration of 1-5 mg/ml.

Fab (antigen binding fragment) of α D11 antibody was generated as previously described (patent application WO05/061540, Covaceuszach et al, 2004). Briefly, Fab fragments were obtained from the corresponding whole monoclonal antibody (IgG form) by proteolysis with papain, followed by an ion exchange chromatography purification step and concentration of the Fab fragments collected in the flow-through. To separate the Fab fragments from the very small amount of uncleaved IgG still present, size exclusion chromatography was performed on a Superdex G75 chromatography column (Pharmacia) using an FPLC system (Pharmacia), followed by a final concentration step.

For both humanized forms of antibodies (Hu- α D11 and Hu-MNAC13) (IgG1/IgG 1/IgG 4), they were also purified as described above, starting from stably transfected cell line supernatants of stable co-transfectants of the heavy chain (pVH/CMVexpress) and light chain (pVL/CMVexpress) of each antibody. The carriers used have been previously disclosed (patent application WO 05/061540). Stably co-transfected clones were obtained by double selection with G418 and mycophenolic acid. To generate the IgG4 variant of Hu- α D11, since the pVH/CMVexpress vector contains a constant portion of human IgG1, it was replaced with the Fc region of the corresponding IgG4 (cloned by RT-PCR from human lymphocyte RNA). IgG1 variants (IgG1 with the N297A mutation, as described by Bolt et al, 1993) were generated by site-directed mutagenesis.

Surface plasmon resonance study

The assay was performed on CM5 chips with amino coupling using a BIAcore 2000 instrument. Coupling was performed using a specific kit purchased from BIAcore, and the coupling reaction was performed according to the manufacturer's instructions.

anti-NGF antibodies were immobilized on the chip and either mouse NGF (m-NGF, Alomone) or recombinant mouse proNGF (rm-proNGF) was injected at decreasing concentrations to obtain binding curves.

Unless otherwise indicated, the flow rate used in the experiments was 30. mu.l/min. The chips were regenerated in all cases with 10mM glycine pH 1.5 (10. mu.L) pulses. The collected data was analyzed using PackageBIAevaluation 3.0. Apparent equilibrium constant K_DDefined as the ka/kd ratio.

Experiments in a mouse pain model

Animals were processed and manipulated according to the guidelines of the IASP ethics Committee and the Italian national method (DL 116/92, application of European Direction 86/609/EEC) for studies using animals. Each necessary attempt was made to minimize pain in the animals and to use the minimum amount of animals required to produce reliable scientific data.

Formaldehyde testing

For the preliminary formaldehyde test (Porro and Cavazzuti, 1993), CD1 male mice (Charles River Labs, Como, Italy) were used weighing 35-40g at the start of the experiment. After their arrival in the laboratory (at least 2 weeks before the experiment), the mice were housed in standard clear plastic cages (4 per cage) at constant temperature (22 ± 1 ℃) and 60% relative humidity, with regular light/darkness (light time from 7 to 19 points). Food and water are not limiting. Experiments were performed between 9 and 14 points. For the formaldehyde test, one animal was placed in a clear plexiglas cage (30X 12X 13cm) and allowed to move freely for 30 minutes before starting the test. After this acclimation period, 20 μ l of formaldehyde solution (5% in saline) was injected subcutaneously (sc) into the back of the right hind paw of the mouse using a micro-syringe with a 26-gauge needle, and observation was started. A mirror is placed behind the cage and a camera is placed in front of the cage to allow unobstructed viewing of the animal's hind paws. The pain index is taken as the licking behavior, i.e. the total amount of time an animal spends licking and/or biting an injected paw. Lick behavior was recorded continuously for 40 minutes and calculated in the continuous 5 minute module (stage 2 corresponds to the 15-40 minute module and may be identified as pain associated with inflammation). In addition, in order to evaluate the effect of formaldehyde injection on spontaneous behaviour, 40 minutes of general behaviour (time spent exploring the environment during walking, standing and leaning) and of self-grooming behaviour (time spent cleaning the face and body) were also continuously recorded during the formaldehyde test. For these parameters, no significant difference was observed after treatment with anti-NGF antibody. In this series of experiments, Fab antibodies (antigen binding fragment, once per antibody: 12.5. mu.g/animal) were administered.

anti-NGF antibody (parent or humanized antibody) or irrelevant Fab was injected subcutaneously (sc) on the back of the right hind paw of each mouse 45 minutes prior to testing using a microsyringe with a 26 gauge needle (injection volume 20 μ l). Each animal was treated only once. And performing blind test on the processing group to which each object belongs. The two stages characteristic of the formaldehyde test were analyzed separately by one-way ANOVA.

Sciatic nerve operation

Male CD1 mice weighing approximately 35g were anesthetized (i.p. injection of 500mg/kg chloral hydrate) to expose the sciatic nerve of the right hind leg, loosely ligated with sewing thread according to the chronic compressive injury model (CCI) of the sciatic nerve as disclosed by Bennett and Xie (1988). Loose ligation at a level above the femur of the sciatic nerve induces peripheral mononeuropathy characterized by thermal/mechanical allodynia and hyperalgesia. Neuropathy developed well 3 days after this injury and persisted for 2-3 months by ligating the nerve at 3 different but close sites.

Pharmacological treatment

Starting on day 3 post-lesion, either the anti-NGF (α D11) blocking antibody or the anti-TrkA (MNAC13) antibody diluted in saline solution (vehicle) was administered in intact form (Mab) as shown in table 1. Either mouse or rat irrelevant immunoglobulin (IgG) at the same dose as the blocking antibody (higher dose if 2 doses were used) or saline solution was used as a control. Each experimental group included 10 animals (unless explicitly indicated otherwise).

Table 1: dosing regimen and determination of mechanical allodynia

Antibodies	Dosage form	I.p. was administered.	Touch pain measurement
				MNAC13αD11	50 ug/mouse-1.4 mg/kg	4 times, on days 3, 4, 5, 6 after injury	Days 3-14
MNAC13αD11	70 ug/mouse 2mg/kg	8 times, on days 3, 4, 5, 6, 7, 8, 9, and 10 after injury	Day 3-31
				MNAC13	30 ug/mouse-0.9 mg/kg

Mechanical allodynia was measured using a plantar dynamic tactile meter (Ugo Basile) as shown in table 1. Day 3 was considered as baseline.

The same protocol was used to evaluate the analgesic effect of the humanized versions of the two antibodies MNAC13 and α D11.

Statistical analysis of the results (CCI experiment)

Results are expressed in two different ways, both as absolute values of threshold pressure values (grams) sufficient to cause the animal to retract the hind legs ipsilateral to the injury, or as percentages of the ratio between the absolute values of the hind legs (ipsilateral/contralateral). The values obtained were statistically analyzed using ANOVA, where the "treatment" factor and the repeated measures (days) were statistically significant and p was < 0.01.

Results

Bonding of

BIACORE studies were performed in order to further identify the binding properties of α D11 anti-NGF antibodies (and humanized variants thereof), identified by assessing the binding affinity of such antibodies to mouse NGF and recombinant mouse pro-NGF. Figure 1 shows the results of these experiments: the α D11 antibody has different reaction kinetics for binding NGF and proNGF. Similar results were obtained with Hu- α D11.

The very small dissociation constant of NGF represents a very tight binding of the antibody to its antigen, a very unique example in antibody binding kinetics. By comparing the binding of anti-NGF antibodies to NGF and to proNGF, it can be assessed that in the latter case the affinity is almost 3 orders of magnitude lower (nanomolar, not picomolar). proNGF is believed to differ from NGF by an additional short amino acid sequence, and this difference in binding affinity is entirely unexpected and surprising.

Since proNGF binds preferentially to p75(Lee, 2001), whereas mature NGF has a higher affinity for the TrkA receptor, α D11 and Hu α D11 can be considered as novel selective inhibitors of TrkA-mediated pathways, particularly relevant for the clinical application of anti-NGF neutralizing antibodies.

Inflammatory pain

The first series of in vivo experiments in mice on formaldehyde-induced pain (inflammatory pain) showed that:

(i) the alpha D11 anti-NGF antibody (Fab form) was shown to be clearly comparable to an unrelated Fab

Significantly reduced pain response (formaldehyde test: stage 2);

(ii) the same results were obtained by replacing α D11 with a humanized variant (Hu- α D11, fig. 2).

This means that Hu- α D11 shows the same potent analgesic properties as α D11 in the relevant inflammatory pain model.

Neuropathic pain

The results of the CCI model showed that the two blocking antibodies MNAC13 and α D11 (fig. 3 and 4) had significant analgesic effects. In particular, similar results were observed for both antibodies at the 1.4mg/kg dose. As shown in fig. 3 and 4, both antibodies had analgesic effects starting on day 2 (day 4) of administration, reaching a maximum effect at about day 6, with substantially the same analgesic efficacy remaining throughout the observation period until day 14. As shown in fig. 4, the results are expressed as a percentage (ratio between the hind paw ipsilateral to the injury and the threshold pressure corresponding to the contralateral hind paw) and may indicate a maximum percentage value of about 60% for each of the two blocking antibodies and about 40% for the control group (IgG and saline).

When the animals were observed for 4 weeks up to day 31, the administration of antibodies blocking the NGF-TrkA system (fig. 5 and 6) indicated a two-stage effect. The first phase of analgesic efficacy (from day 3 to day 17, i.e. until one week after the last injection) was characterized by a maximal effect at about days 11-12. After a decrease in action (up to day 17), an increase in second phase analgesia up to day 31 was observed. It is therefore possible to distinguish between the two phases of the analgesic effect of NGF/TrkA blocking antibodies: a first phase (pharmacological effect) comprising the treatment period and the first week after the last injection of antibody (the week during which the effect is reduced, consistent with the blood concentration of antibody); the second phase is a long-term effect, possibly requiring new gene expression, which makes these antibodies unique to the "disease-modulating" active principle (in the field of neuropathic pain), i.e. capable of deeply modulating the disease course, unlike the simple pharmacological effects on symptoms of the products commonly used in this treatment. In FIG. 5, the analgesic effect of 2 doses of MNAC13 anti-TrkA (2 and 0.9mg/Kg) and α D11(2mg/Kg) are compared. The results are expressed as percentages. The time pattern of α D11 efficacy was similar to MNAC13, although animals treated with α D11 were indistinguishable from the control group (IgG) on day 17, while those treated with MNAC13 were still significantly different (p < 0.01). From day 21, α D11 restored analgesia to a final level similar to MNAC13 (greater than 60%, compared to control 40%) on day 31.

When the humanized form variant (Hu- α D11) thereof was used instead of the α D11 antibody, substantially the same results as above were obtained (the dose used for each antibody was 2mg/kg), confirming that the humanized form of the antibody had the same analgesic effect as the parent form thereof. The antibodies were humanized in the light chain (SEQ ID No.3) and heavy chain (SEQ ID No.4) variable regions using the methods described in WO 2005/061540. To construct fully humanized antibodies, different constant regions (SEQ ID Nos. 5-8) as described above were used.

As a representative example of equivalents of analgesic activity (CCI) of the parent and humanized antibodies, FIG. 6 shows a comparison between α D11 and Hu- α D11(IgG4 format).

Based on this, it can be shown that Hu- α D11 has the same long-term effect as its parent form.

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Claims (13)

1. Use of a human or humanized anti-NGF antibody capable of inhibiting the binding between NGF and TrkA, wherein the antibody is an IgG4 isotype antibody and the antibody light chain variable region comprises three Complementarity Determining Regions (CDRs) consisting of the sequences of amino acids 24-34 of SEQ ID No.1, amino acids 50-56 of SEQ ID No.1 and amino acids 89-97 of SEQ ID No.1, and the antibody heavy chain variable region comprises three Complementarity Determining Regions (CDRs) consisting of the sequences of amino acids 26-35 of SEQ ID No.2, amino acids 50-65 of SEQ ID No.2 and amino acids 98-111 of SEQ ID No.2, in the manufacture of a medicament for the treatment and/or prevention of chronic pain.

2. The use of claim 1, wherein the antibody comprises two light chains and two heavy chains.

3. Use according to claim 1, wherein the light chain variable region consists of the sequence shown in SEQ ID No. 3.

4. Use according to claim 1, wherein the heavy chain variable region consists of the sequence shown in SEQ ID No. 4.

5. Use according to claim 1, wherein the light chain consists of the sequence shown in SEQ ID No. 8.

6. Use according to claim 1, wherein the heavy chain consists of the sequence of SEQ ID No. 7.

7. Use according to claim 1, wherein said light chain consists of the sequence shown in SEQ ID No.8 and said heavy chain consists of the sequence shown in SEQ ID No. 7.

8. The use of claim 1, wherein the pain is chronic inflammatory pain.

9. The use of claim 8, wherein the chronic pain is due to: pancreatitis, kidney stones, headache, dysmenorrhea, musculoskeletal pain, sprain, visceral pain, ovarian cyst, prostatitis, cystitis, interstitial cystitis, post-operative pain, migraine, trigeminal neuralgia, pain from burns and/or wounds, pain associated with trauma, neuropathic pain, pain associated with musculoskeletal diseases, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, periarticular lesions, neoplastic pain, bone metastasis pain, HIV pain.

10. The use of claim 9, wherein the pain is neuropathic pain.

11. The use of claim 9, wherein the pain is neoplastic pain.

12. The use of claim 1, wherein the antibody has a long-term effect.

13. A kit comprising an antibody as defined in claim 1 and instructions for administering the composition to a subject in need of treatment for chronic pain, thereby treating chronic pain in the subject.